Genome-wide characterization of microsatellites in cobia Rachycentron canadum (Linnaeus, 1766): Survey and analysis of their abundance and diversity.

The cobia Rachycentron canadum, mainly distributed in the warm waters of tropical and subtropical regions around the world, remains a fish of considerable economic importance. Detailed diversity and the number of microsatellite sequences in the cobia genome are still unintelligible. The primary aim of this work was to identify and quantify the miscellaneous SSR sequences in the cobia genome. More than 280,000 sequences were sequenced and screened using next-generation sequencing technology and microsatellite identification. Perfect mononucleotide repeats, dinucleotide microsatellites, and trinucleotide microsatellites contain (A)10 /(T)10 , (AC)6 /(TG)6 , and (AAT)5-32 as the largest number of motifs in each type of microsatellite, respectively. The tetranucleotide and pentanucleotide microsatellites (TTM and PTM) consist of the largest number of motifs of both (ATCT)5-32 and (TCAT)5-31 in TTMs, and (CTCTC)5-9 in PTMs, whereas the hexanucleotide microsatellites are rarely observed in the cobia genome. All c. 38000 sequences of composite microsatellites are extremely diverse, including compound (11.71%), interrupted compound (71.77%), complex (0.45%), and interrupted complex (16.07%). In this study, we developed a convenient and useful recording system for writing down and categorizing diverse composite microsatellite types. This system will provide great support for exploring repeat origins, evolutionary mechanisms, and the application of polymorphic microsatellites.

A SNP-Based Genome-Wide Association Study to Mine Genetic Loci Associated to Salinity Tolerance in Mungbean (Vigna radiata L.).

Mungbean (Vigna radiata (L.) R. Wilzeck var. radiata) is a protein-rich short-duration legume that fits well as a rotation crop into major cereal production systems of East and South-East Asia. Salinity stress in arid areas affects mungbean, being more of a glycophyte than cereals. A significant portion of the global arable land is either salt or sodium affected. Thus, studies to understand and improve salt-stress tolerance are imminent. Here, we conducted a genome-wide association study (GWAS) to mine genomic loci underlying salt-stress tolerance during seed germination of mungbean. The World Vegetable Center (WorldVeg) mungbean minicore collection representing the diversity of mungbean germplasm was utilized as the study panel and variation for salt stress tolerance was found in this germplasm collection. The germplasm panel was classed into two agro-climatic groups and showed significant differences in their germination abilities under salt stress. A total of 5288 SNP markers obtained through genotyping-by-sequencing (GBS) were used to mine alleles associated with salt stress tolerance. Associated SNPs were identified on chromosomes 7 and 9. The associated region at chromosome 7 (position 2,696,072 to 2,809,200 bp) contains the gene Vradi07g01630, which was annotated as the ammonium transport protein (AMT). The associated region in chromosome 9 (position 19,390,227 bp to 20,321,817 bp) contained the genes Vradi09g09510 and Vradi09g09600, annotated as OsGrx_S16-glutaredoxin subgroup II and dnaJ domain proteins respectively. These proteins were reported to have functions related to salt-stress tolerance.

Variation in the Karyotype, Cytochrome b Gene, and 5S rDNA of Four Thunnus (Perciformes, Scombridae) Tunas.

Yan-Horn Lee, Tsair-Bor Yen, Chiu-Fen Chen, and Mei-Chen Tseng (2018) Thunnus tunas in Scombridae are divided into the temperate subgenus Thunnus (bluefin group) and tropical subgenus Neothunnus (yellowfin group) species based on anatomic traits and distributions. The main purpose of this study was to examine the systematic status of T. obesus based on karyotype, cytochrome (Cyt) b gene, and 5S ribosomal DNA sequences. All T. obesus, T. albacares, T. alalunga, and T. orientalis specimens were caught in southeastern coastal waters off the main island of Taiwan. The karyotypical formula of T. obesus was 2 m + 2 st + 44 t, that of T. albacares was 2 m + 2 sm + 2 st + 42 t, that of T. alalunga was 2 m + 2 sm + 2 st + 42 t, and that of T. orientalis was 2 m + 2 sm + 44 t (m: metacentric; sm: submetacentric; st: subtelocentric; t: telocentric chromosome). According to a molecular genetics analysis for these species using Cyt b gene sequences (1141 bp), interspecific genetic distances ranged from 0.004 (T. orientalis vs. T. alalunga) to 0.038 (T. alalunga vs. T. obesus). The genealogy tree exhibited these 4 species as being categorized into 4 monophyletic groups with high bootstrapping values; T. alalunga and T. orientalis are sister species. This result suggests that the species currently allocated in Thunnus and Neothunnus might need new taxonomic characters to redefine the monophyly of the two subgenera. The sequence lengths of all cloned 5S genes from the 4 species ranged from 327-342 bp. Interspecific genetic distances ranged from 0.016 (T. orientalis vs. T. alalunga) to 0.111 (T. orientalis vs. T. albacares). The phylogenetic tree based on 5S rDNA shows T. obesus divided into 2 groups: one similar to T. albacares and the other close to T. orientalis. These results imply that Thunnus tunas have a common synapomorphic character with Scombridae fish (2n = 48) and high numbers of telocentric chromosomes (42-44). Thunnus orientalis and T. alalunga are sister based on molecular data. Thunnus obesus may have been derived from a more-complicated speciation processes.

Comparative Study on Hatching Rate, Survival Rate, and Feminization of Onychostoma barbatulum (Pellegrin, 1908) at Different Temperatures and Examining Sex Change by Gonad and Karyotype Analyses.

Mei-Chen Tseng, Dian-Hao Yang, and Tsair-Bor Yen (2017) Onychostoma barbatulum has become an aquaculture species with high economic value in Taiwan. It was observed that females can grow faster to a larger size than males on aquaculture farms. Therefore, development of feminized fry can increase farm profits in the future. The purpose of this study was to establish the optimal incubation, feed, and feminization temperatures to produce a high feminization ratio and better survival rates for O. barbatulum fry. The performance mode of sex-determination by temperature was also explored in the study. Adults were collected from Nanzixian Stream in southwestern Taiwan and artificially propagated in tanks. Fertilized eggs were placed in environments of different temperatures to determine the hatching rate (n = 3000) and the survival rate (n = 3000) of fry. After 6 months, the sex ratio was established from gonad tableting (n = 360). A karyotype analysis (n = 86) was also conducted to verify the existence of gender-reversed individuals. The results showed that hatching rates at 17.5, 19.5, 21.5, 23.5, and 25.5°C were 70.7%, 67.3%, 73.3%, 33.7%, and 34.7%, respectively. Survival ratios from low to high temperatures were 34.7%, 47.7%, 33.7%, 12.3%, and 23.3%, respectively. These results indicated that both the hatching rates and survival ratios of fry performed poorly at temperatures higher than 21.5°C. The performance mode of sex-determination by temperature of O. barbatulum revealed that female ratios significantly decreased at the two extremes of the temperature range, while the female ratio was highest at an intermediate temperature. The best feminized ratio (83.4%) was observed at 21.5°C among all tested temperatures (p < 0.05). Meanwhile according to a karyotype analysis, sex-reversed individuals were found in each group, indicating that temperature is a critical phenotypic sex-determining factor. Therefore, rearing O. barbatulum fry at a proper water temperature can effectively increase the female sex ratio and maintain high hatchability and survival ratios. These results can potentially be applied to produce a high proportion of female fry on aquaculture farms.

Cytogenetics of Two Onychostoma Species in Taiwan by Ag-NOR and 18S rDNA Profiles.

Chiao-Chuan Han, Tsair-Bor Yen, Nian-Cih Chen, and Mei-Chen Tseng (2017) Both Onychostoma barbatulum and O. alticorpus are primary freshwater fish in Taiwan. The former has been developed as an aquaculture species with high economic value, while the latter is a native endemic species in Taiwan. Understanding the cytogenetic information of these two species is necessary for their selected breeding, recovery, and management. In this study, Giemsa staining, silver-binding nucleolar organizer region (Ag-NOR), C-banding, and fluorescence in situ hybridization (FISH) with 18S ribosomal (r)DNA probes were used to analyze the cytogenetic characteristics. Results of Giemsa staining showed that the two Onychostoma species shared the same number of chromosomes, 2n = 50. Respective karyotype formulas of the female and male were 10 m + 22 sm + 10 st + 8 t and 11 m + 22 sm + 10 st + 7 t in O. barbatulum, and 14 m + 18 sm + 8 st + 10 t and 15 m + 18 sm + 8 st + 9 t in O. alticorpus. Karyotypes of both species showed a pair of heteromorphic chromosomes in male fish. Their sex determination should be the XX/XY system. Two pairs of Ag-NORs were found in O. barbatulum, but only one pair occurred in O. alticorpus. C-banding areas were observed on centromeres or telomeres of some chromosomes. FISH revealed different cytogenetic characters between these two species. The above cytogenetic information will contribute to species identification, population recovery, and advantages for breeding and management in the future.

Comparative Studies of 5S rDNA Profiles and Cyt b Sequences in two Onychostoma Species (Cyprinidae).

Onychostoma barbatulum and O. alticorpus, two primarily freshwater cyprinid fish, have similar morphological characters and partially overlapping ecological habitats. In order to explore the genetic differences between these two species, chromosomal characteristics and genetic variations were examined by fluorescence in situ hybridization (FISH) of 5S rDNA and cytochrome (Cyt) b gene analysis. Ten specimens of O. barbatulum and O. alticorpus were collected from the Nanzihsian Stream in southern Taiwan. FISH revealed that the 5S rDNA loci of O. barbatulum and O. alticorpus were found at a pericentromeric and subtelomeric position, respectively, in a pair of submetacentric chromosomes. Cyt b genes were amplified and sequenced from five individuals of each species. Intraspecific genetic distances ranged from 0.001-0.004 in O. barbatulum and from 0.001-0.006 in O. alticorpus. Genetic distances between these two species ranged from 0.132-0.142. The phylogenetic tree showed these two species are not sister species. In conclusion, FISH cytogenetic information and Cyt b gene analyses indicated that these two species have significantly different genetic characteristics; nevertheless, their morphological similarities may be due to environmental adaptation.

Synergistic effects of cinnamaldehyde in combination with eugenol against wood decay fungi.

The combined effects of using cinnamaldehyde with catechin, quercetin or eugenol against wood decay fungi were examined by comparing their isoeffective concentrations to that of individual compound. Among all combinations, cinnamaldehyde with eugenol revealed the strongest synergy against Laetiporus sulphureus. The synergism was due to the interference of fungal cell wall synthesis and cell wall destruction plus radical scavenging effect. Results also suggested that antioxidant with fungicidal effect might be a better candidate than pure antioxidant for the system of fungicide/antioxidant.

Antifungal properties of ethanolic extract and its active compounds from Calocedrus macrolepis var. formosana (Florin) heartwood.

The ethanolic extract of Calocedrus macrolepis var. formosana heartwood was screened for antifungal compounds by agar dilution assay and liquid chromatography. Two compounds, beta-thujaplicin and gamma-thujaplicin, responsible for the antifungal property of C. macrolepis var. formosana heartwood were isolated by high performance liquid chromatography (HPLC), and identified by 1H NMR and 13C NMR. The antifungal activities of these two compounds were further evaluated against total 15 fungi, including wood decay fungi, tree pathogenic fungi and molds. The hexane soluble fraction showed the strongest antifungal activities among all fractions. beta-Thujaplicin and gamma-thujaplicin exhibited not only very strong antifungal activity, but also broad antifungal spectrum. The MIC values of beta-thujaplicin and gamma-thujaplicin were in the range of 5.0-50.0 microg/ml. In addition, scanning electron microscopy (SEM) was carried out to study the structural change of fungal hyphae induced by beta-thujaplicin. Strong cell wall shrinkage indicated the fungicidal effect could be attributed to the combined actions of metal chelating and cytoplasm leakage. It also suggests that the role of metal chelating is indispensable in the design of environmental-friendly fungicides.